Silvia Cermelli

Parathyroid Hormone [PTH(1–34)] and Parathyroid Hormone–Related Protein [PTHrP(1–34)] Promote Reversion of Hypertrophic Chondrocytes to a Prehypertrophic Proliferating Phenotype and Prevent Terminal Differentiation of Osteoblast‐like Cells

The effects of parathyroid hormone/parathyroid hormone–related protein (PTH/PTHrP) on late events in chondrocyte differentiation were investigated by a dual in vitro model where conditions of suspension versus adhesion culturing are permissive either for apoptosis or for the further differentiation of hypertrophic chondrocytes to osteoblast‐ like cells. Chick embryo hypertrophic chondrocytes maintained in suspension synthesized type II and type X collagen and organized their extracellular matrix, forming a tissue highly reminiscent of true cartilage, which eventually mineralized. The formation of mineralized cartilage was associated with the expression of alkaline phosphatase (ALP), arrest of cell growth, and apoptosis, as observed in growth plates in vivo. In this system, PTH/PTHrP was found to repress type X collagen synthesis, ALP expression, and cartilage matrix mineralization. Cell proliferation was resumed, whereas apoptosis was blocked. Hypertrophic chondrocytes cultured in adherent conditions in the presence of retinoic acid underwent further differentiation to osteoblast‐like cells (i.e., they resumed cell proliferation, switched to type I collagen synthesis, and produced a mineralizing bone‐like matrix). In this system, PTH addition to culture completely inhibited the expression of ALP and matrix mineralization, whereas cell proliferation and expression of type I collagen were not affected. These data indicate that PTH/PTHrP inhibit both the mineralization of a cartilage‐like matrix and apoptosis (mimicked in the suspension culture) and the production of a mineralizing bone‐like matrix, characterizing further differentiation of hypertrophic chondrocytes to osteoblasts like cells (mimicked in adhesion culture). Treatment of chondrocyte cultures with PTH/PTHrP reverts cultured cells in states of differentiation earlier than hypertrophic chondrocytes (suspension), or earlier than mineralizing osteoblast‐like cells (adhesion). However, withdrawal of hormonal stimulation redirects cells toward their distinct, microenvironment‐dependent, terminal differentiation and fate.

The Developmentally Regulated Avian Ch21 Lipocalin Is an Extracellular Fatty Acid-binding Protein

Ch21, a developmentally regulated extracellular protein expressed in chick embryos and in cultured chondrocytes, was expressed in the baculovirus system, and the recombinant protein was purified to homogeneity by gel-filtration chromatography. Separation of two isoforms was achieved on an ion-exchange column. Previous work had shown that Ch21 belongs to the superfamily of lipocalins, which are transport proteins for small hydrophobic molecules. Studies were performed to identify the Ch21 ligand. By analysis of recombinant Ch21 on native polyacrylamide gel electrophoresis and by Lipidex assay, the binding of fatty acid to the protein was shown and a preferential binding of long-chain unsaturated fatty acids was observed. Both isoforms had the same behavior. The binding was saturable. Stoichiometry was about 0.7 mol of ligand/mol of protein. The protein binds the ligand in its monomeric form. Calculated dissociation constants were 2 × 10−7 M for unsaturated fatty acids and 5 × 10−7 M for stearic acid. The binding was specific; other hydrophobic molecules, as retinoic acid, progesterone, prostaglandins, and long-chain alcohols and aldehydes did not bind to the protein. Short-chain fatty acids did not bind to the protein. Ch21, also present in chicken serum, represents the first extracellular protein able to selectively bind and transport fatty acid in extracellular fluids and serum. We propose to rename the Ch21 protein as extracellular fatty acid-binding rotein (Ex-FABP).

Ex-FABP: A fatty acid binding lipocalin developmentally regulated in chicken endochondral bone formation and myogenesis

Extracellular fatty acid binding protein (Ex-FABP) is a 21 kDa lipocalin specifically binding fatty acids, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibers and in blood granulocytes. In chondrocyte and myoblast cultures Ex-FABP expression is increased by inflammatory agents and repressed by anti-inflammatory agents. In adult cartilage Ex-FABP is expressed only in pathological conditions such as in dyschondroplastic and osteoarthritic chickens. The possible mammalian counterpart is the Neu-related lipocalin (NRL), a lipocalin overexpressed in rat mammary cancer; NRL is homologous to the human neutrophil gelatinase associated lipocalin (NGAL) expressed in granulocytes and in epithelial cells in inflammation and malignancy and to the Sip24 (super-inducible protein 24), an acute phase lipocalin expressed in mouse after turpentine injection. Immunolocalization and in situ hybridization showed that NRL/NGAL is expressed in hypertrophic cartilage, in forming skeletal muscle fibers and in developing heart. In adult cartilage NRL/NGAL was expressed in articular cartilage from osteoarthritic patients and in chondrosarcoma. Moreover, NRL was induced in chondrocyte and myoblast cultures by an inflammatory agent. We propose that these lipocalins (Ex-FABP, NRL/NGAL, Sip24) represent stress proteins physiologically expressed in tissues where active remodeling is taking place during development and also present in tissues characterized by an acute phase response due to pathological conditions.

Ex-FABP, extracellular fatty acid binding protein, is a stress lipocalin expressed during chicken embryo development

Extracellular Fatty Acid Binding Protein (Ex-FABP) is a 21 kDa lipocalin, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibres and in blood granulocyte. The protein selectively binds with high affinity fatty acids, preferably long chain unsaturated fatty acids in chondrocyte and myoblast cultures Ex-FABP expression is increased by inflammatory-agents and repressed by anti-inflammatory-agents. In adult cartilage, Ex-FABP is expressed only in pathological conditions such as in dyschondroplastic and osteoarthritic chicken cartilage. We propose that lipocalin Ex-FABP represents a stress protein physiologically expressed in tissues where active remodelling is taking place during development and also present in tissues characterized by a stress response due to pathological conditions.

Expression of the extracellular fatty acid-binding protein during muscle fiber formationin Vivo andin Vitro

Extracellular fatty acid-binding protein (Ex-FABP) is a 21-kDa protein developmentally regulated in chick embryo, first observed in vitro in chondrocytes at a late stage of differentiation as a protein secreted into the culture medium (1). Immunolocalization performed in cartilage tissues with polyclonal antibodies obtained against the purified protein showed that the protein is present in vivo in chicken embryo tibia in hypertrophic chondrocytes of the growth plate at the border between hypertrophic cartilage and newly forrned bone, in the cartilage surrounding marrow cavities and blood vessels, and in the epiphysial prearticular cartilage. The complete amino acid sequence of the protein, obtained by combining protein and nucleotide sequence data, assigned the protein to the superfamity of lipocalins or lipophitic molecules carrier proteins (2). The recombinant protein was obtained in the baculovirus system, and studies were performed to identify a possible ligand. By lipidex binding assay with radioactive lipophilic compounds, a specific fatty acid binding to the protein was shown and a preferential binding of long-chain unsaturated fatty acids was observed (3). Calculated dissociation constant was 2 x 10-7M for unsaturated fatty acids and 5 x 10 .7 M for stearic acid. Shortchain fatty acids did not bind to the protein. Other hydrophobic molecules as retinoic acid, retinol, progesterone, prostaglandins, and long-chain alcohols and aldehydes did not bind to the protein. It is widely accepted that free fatty acids in blood are transported by albumin. Ex-FABP is present in chicken serum and represents the first extracellular protein able to selectively bind and transport fatty acids in extracellular fluid and serum. Ex-FABP could represent inblood the high-affinity, low-capacity, specific binding protein that transports fatty acids to target organs while albumin could represent a tow-affinity, high-capacity storage protein for fatty acids. Ex-FABP is also produced by peripheric granutocytes. We performed studies aimed to elucidate a possible role of the protein during chicken embryo development, and we observed that Ex-FABP is expressed in the forming myotubes both in vitro and in vivo. The presence of the protein and of the mRNA was observed in newly formed myotubes at early stages of chick embryo development by im-